1. Field of the Invention
The invention relates generally to the field of electrochemical sensor technology and specifically to sensors that utilize colloidal gold to detect metals and to methods of using the sensors for small volume determination of submicromolar levels of these analytes.
2. Description of the Related Art
Lead is a toxic heavy metal that affects all body systems and is ubiquitous in industrialized society. Children are especially at risk. It is estimated that in the United States 3 million out of a total population of 24 million children under the age of six have lead poisoning. It is reported that blood lead as low as 10 .mu.g/dL is responsible for adverse effects on intelligence and behavior with negative endocrinological and hematological impacts. The U.S. Centers for Disease Control (CDC) has set the blood lead action level at 10 .mu.g/dL for children under six and recommends that this population be screen-tested annually. There is a need for new, portable, low-cost, technology to meet this goal.
CDC's identification of the magnitude of this problem has created a demand for a portable instrument capable of detecting low blood-lead and low environmental-lead levels. A simple to use easily transported lead measuring device would be particularly useful in remote locations where clinics and doctors offices are not accessible to the population and where highly trained technical personnel are not available.
Increasingly, undesirable chemicals are released into the environment as a result of manufacturing processes and waste disposal activity. Unfortunately, these substances leach into soil, into water supplies, and eventually into the food chain. The ideal solution is to prevent pollution at the source; even so, a first consideration is to determine whether or not an undesirable compound is present in a specific location.
The measurement of lead in solution by stripping analysis is a well-established technique. Traditionally, this technique has relied on laboratory based instrumentation, skilled operators, reusable electrodes, and the use of mercury metal. The most popular currently used technique for blood-lead measurement is anodic stripping voltammetry (ASV) utilizing reusable large area graphite-mercury composite electrodes. However, this technology is complicated, expensive for routine use, and relatively insensitive. Additionally, the use of mercury has several drawbacks, including volatility and toxicity. Mercury in any form is toxic. Its effects are particularly insidious because toxicity is cumulative, building up after successive exposures to even relatively small amounts of the metal. Because mercury is a liquid, it requires special precautions and considerations in handling.
The testing procedures presently available are not suitable to meet the public demand for mass blood-lead screening. Because ASV utilizes reusable electrodes, the technique requires a well-trained and experienced analytical chemist to conduct a reliable electrochemical measurement. This is because the electrode must be restored and prepared for each analytical session.
There is therefore a need to develop rapid, simple and reliable tests for determining trace amounts of different classes of materials, especially molecular entities such as trace elements and contaminants. Furthermore, a means of simple, reliable and rapid on-site testing is needed to detect such molecules in remote or non-institutional locations in the field, in small clinics, doctors' offices, or even in the home. A particularly useful application includes testing body-fluid samples and environmental samples for trace elements by relatively unskilled personnel.
Likewise, a simple, rapid, disposable, and inexpensive analyte detection system would greatly benefit developing countries that may lack access to the sophisticated equipment, facilities and trained personnel necessary for traditional testing methods. Finally, methods are needed to detect trace levels of analytes, so that effective, point source detection of environmental pollutants and areas of contamination can be more easily identified.
Traditional stripping voltammetry is performed primarily with mercury or mercury modified electrodes because mercury affords easy surface regeneration and formation of alloys to prevent surface change or fouling. However, mercury is toxic and mercury or mercury modified electrodes are not stable for repeated or continuous monitoring.
Certain gold surfaces do form alloys with metals, although the capacity may not be as great as mercury film. However, a gold film is much more stable than a mercury film electrode and the formed gold alloy can be restored to the original gold film electrode electrochemically after the stripping. This indicates the feasibility of using gold based electrodes for continuous monitoring of some heavy metals through selected amperometric processes.
Coulometry and other electrochemical techniques have been applied to the analysis of metals at polycrystalline solid electrodes (Lord et al., 1952; Nicholson, 1957; Nicholson, 1960; Hamelin, 1979; Wang and Tian, 1993; Wojciechowski and Balcerzak, 1990). Quantitation of deposition and dissolution of metals (stripping analysis) on solid electrodes such as Pt or Au has advantages in the possibility to analyze metals more electropositive than mercury, and in greater sensitivity because deposited metal is recovered more completely (Nicholson, 1957).
While platinum (Pt) has been employed for stripping analysis of the greatest number of metals (Lord et al., 1952; Nicholson, 1957; Nicholson, 1960), gold (Au) has been employed for trace analysis of lead in aqueous solution (Wang and Tian, 1993; Wang, 1985; Copeland and Skogerboe, 1974; Posey and Andrew, 1980). Lead in dilute acid solution has been detected and results shown analytically useful in deposition at Au films (Wang and Tian, 1993) and Pt wires (Lord et al., 1952). On Au films plated on carbon substrates, lead has been detected to 2.5 ppb (12.5 nM) (Wang and Tian, 1993). Similar results for analysis of nickel (Ni) at Pt and Au have been shown,.suggesting that extension of analysis from one noble metal to another as a stripping substrate is reasonable (Nicholson, 1957).
Hydrogen evolution on gold in aqueous solution is suppressed by deposition of even submonolayer coverages of lead (Hamelin, 1979), but dissolved oxygen may interfere in voltammetric analysis of trace metal levels-in stripping at Au or Pt (Nicholson, 1960). While lead on Au at submonolayer coverages shows no alloy formation (Hamelin, 1979), higher coverages (bulk deposits) may yield alloys (Biberian and Rhead, 1973; Perdereau et al., 1974).
Deposition of lead occurs on Pt or Au in the range of potentials -0.55 V to -0.7 V (vs. S.C.E.). Solution conditions range from pH 2 (0.01M HClO4) to pH .about.5 (0.1M KCl). Depositions have been employed in stirred or unstirred solution, for times ranging from 0.5 to 10 minutes (or greater for unstirred solutions of trace levels of metal). Scans through the dissolution potential range yield peaks with FWHM&lt;100 mV (on Pt) (Lord et al., 1952) and Au (Wang and Tian, 1993). Cleaning of the surface, when necessary, has been accomplished by formation and reduction of Au oxide (Hamelin, 1979).
Monodisperse colloidal gold particles having diameters ranging from 50 to 1,000 .ANG. have a large surface area per unit volume and have found extensive use as markers in electron microscopy (Housberger, 1981). Colloidal gold particles have been labeled by the adsorption of a variety of biological macromolecules, including toxins, antibodies, proteins, and enzymes (BBI International). Gold is also an excellent electrode material with good heterogeneous electron transfer characteristics (Sawyer and Roberts, Jr., 1974).